ES2373075T3 - PROCEDURE TO INCREASE THE ACTIVITY OF ASPARAGINASE IN A SOLUTION. - Google Patents

Abstract

Method for increasing the activity of asparaginase in a solution, said procedure comprising the steps consisting of: a) treating a potable water to prepare a treated water, wherein said treated water further comprises a chlorine concentration of less than about 0, 5 ppm; and b) mixing the asparaginase with said treated water to prepare an asparaginase solution.

Description

Procedure to increase the activity of asparaginase in a solution.

Background of the invention

Technical field

The present invention relates to a process for reducing the amount of asparagine, a precursor of acrylamide, in a food product. More particularly, the present invention relates to increasing the stability of the asparaginase enzyme in a solution.

Description of the related technique

As set forth in US Patent No. 7,037,540, acrylamide has been found in thermally processed foods containing asparagine. The level of acrylamide formed in some food products can be reduced by adding the asparaginase enzyme to the food product before cooking.

The addition of acrylamide reducing enzymes, such as asparaginase, to food products on a commercial scale, and not on a batch scale, presents several difficulties. For example, the enzyme asparaginase should contact free asparagine to facilitate its hydrolysis. Because the enzyme is typically supplied in a relatively concentrated form, the enzyme is ideally mixed and diluted in an aqueous base solution before contacting the food product with the enzyme solution. For example, contacting the food product with the enzyme solution may comprise the formation of a dough and the mixing of an enzyme solution with the dough.

A known way to quantify the activity of an enzyme is expression in terms of units. A unit of enzymatic activity is defined as the amount of enzyme needed as a catalyst to convert a substrate micromol in one minute. Thus, from the knowledge of the relative concentration of substrate or compound, such as asparagine, in a food product and the amount thereof, the number of enzyme units, such as asparaginase, necessary to convert the chemical compound can be calculated. desired, in this case asparagine, in a different chemical compound.

For reasons unknown above, even in the case that excess doses are used (that is, an amount exceeding the mathematically expected amount necessary to convert all asparagine into the food product) of the enzyme asparaginase into a food product, such as mashed potatoes. Potatoes or corn dough, measurable levels of asparagine are often observed in the dough. Because it is desired to reduce the level of acrylamide formed by thermally processing certain foods, it would be desirable to have a system and procedure for maximizing the effectiveness of an enzyme used to reduce acrylamide precursors in commercially prepared food products.

CN No. 1137064 discloses a process for preparing an aqueous solution rich in asparaginase, including the 4-stage fermentation of lean meat and sterilization.

JP No. 6030782 describes a procedure for the treatment of wastewater.

Summary of the invention

A first aspect of the present invention provides a process for increasing the activity of asparaginase in a solution, the process being as defined in claim 1.

In some embodiments, chlorine is removed by ion exchange, reverse osmosis, activated carbon and / or by air bubbling. In some embodiments, additives such as reducing agents and chlorine sequestrants are used to treat drinking water.

A second aspect of the present invention provides a system as defined in claim 11.

The foregoing, as well as the additional features and advantages of the present invention, will be apparent from the detailed description provided below.

Brief description of the drawings

The new features believed to be typical of the invention are set forth in the appended claims. However, the invention itself, as well as a preferred mode of use, and the additional objectives and advantages thereof, will become more clearly apparent by reference to the following detailed description of the illustrative embodiments from the accompanying drawings , in which:

Figure 1a is a graphic representation of the residual enzymatic activity after various drinking water treatments, and

Figure 1b is a graphical representation of the residual enzymatic activity of various mixtures of saline water.

Detailed description

In one embodiment, the present invention relates to how to provide an aqueous-based solution that increases asparaginase stability and preserves asparaginase activity. The increased activity of asparaginase can result in a more effective reduction of acrylamide in food products because asparagine is a precursor to acrylamide. As used herein, the expression "enzymatic activity" It is expressed in units. Each unit of asparaginase can hydrolyze a micromol of asparagine in one minute.

In one embodiment, the food product in which it is desired to reduce the level of acrylamide formed during thermal processing is formed from a dough. The expression " manufactured snack " It refers to a snack food that uses as its starting ingredient something different from the original and unaltered feculent starting material. For example, manufactured snacks include manufactured chips that use a dehydrated potato product as a starting material, and corn potatoes that use dough flour as their starting material. It should be appreciated herein that the dehydrated potato product may be potato flour, potato flakes, potato granules or other forms in which dehydrated potatoes are present. In the use of any of these expressions in the present application, it should be appreciated that all such variations are included. Only by way of non-limiting example, the examples of "manufactured foods" to which an asparaginase solution can be added include tortillas, corn potatoes, chips prepared from potato flakes and / or mashed potatoes, multigrain potatoes, puffed corn, puffed wheat, puffed rice, toast, bakery (such such as rye flour, wheat, oatmeal, potato, white, whole grain and mixed), soft and hard salted donuts (" pretzels "), cupcakes, cookies, toast, corn pancakes, flour pancakes, pita bread , croissants, puff pastry, muffins, chocolate and nut biscuits ("brownies"), cakes, donuts ("bagels"), donuts, cereals, extruded snacks, granola products, flours, cornmeal, dough, flakes of potato, polenta, mixes for breading and pastry products, chilled and frozen doughs, reconstituted foods, processed and frozen foods, breaded meats and vegetables, golden potatoes, mashed potatoes, crepes, pancakes, waffles, dough Pizza, peanut butter, foods containing chopped and processed nuts, jellies, fillings, fruit paste, mashed vegetables, alcoholic beverages such as beers and ales, cocoa, cocoa powder, chocolate, hot chocolate, cheese, animal foods such as feed for dogs and cats, and any other human or animal food product that is subjected to rolling or extrusion or is prepared from a dough or mixture of ingredients.

The use of the expression "manufactured foods" herein includes refreshments manufactured as defined above. The use of the expression "food products" herein includes all manufactured snacks and manufactured foods as defined above.

As referenced herein, thermally processed foods comprise foods that can be treated with an asparaginase solution, by way of non-limiting example, all foods presented above as examples of manufactured snacks and manufactured foods, as well as chips , sliced potatoes, sweet potato slices, other tuber or root materials, cooked vegetables, including asparagus, cooked onions and tomatoes, coffee beans, cocoa beans, cooked meats, dehydrated fruits and vegetables, heat-processed animal feed, tobacco , tea, roasted or cooked nuts, soybeans, molasses, sauces such as barbecue sauce, slices of banana, apple slices, fried plantains and other cooked fruits.

According to some of said embodiments, the desired ingredients for preparing the dough are mixed with water and the desired amount of asparaginase is also mixed with treated water in order to prepare the asparaginase solution. The asparaginase solution can then be added to the dough. In one embodiment, an asparaginase solution is mixed directly with the desired ingredients, preparing a dough. The dough can then be prepared in a thermally processed food product.

In commercial facilities, the water used to form the mass and the asparaginase solution is the water that can be easily disposed in the facilities, which is typically potable water supplied to the end user by the local municipal water supplier. As used herein, "drinking water" It refers to the water supplied by a drinking water supplier, and includes, in a non-limiting manner, water from a municipal water supplier. Virtually all municipal water suppliers in the United States add enough chlorine to drinking water so that drinking water has residual chlorine when it reaches the customer's tap. Many municipal water supply companies add chloramine to drinking water because chloramine is more stable than chlorine. As used herein, chlorine is defined as the oxidizing forms of chlorine and includes, but is not limited to, chloramine and hypochlorites. Similarly, non-oxidizing forms of the chlorine ion, such as those provided by hydrochloric acid (HCl) and sodium chloride (NaCl) are not included in the definition.

It has been found that certain characteristics of drinking water, for example the presence of chlorine, reduce the

5 asparaginase enzyme activity to a point where it is not useful in a commercial context for food production. As used herein, "residual enzyme activity" (expressed in%) refers to the enzymatic activity of a control divided by the enzymatic activity of a sample, and provides a relative measure of the enzymatic activity under various experimental conditions. Procedures and systems to mitigate the effect of drinking water on the activity have also been identified.

10 enzymatic and preserve the residual enzymatic activity of asparaginase so that it can be useful in a commercial context. The following examples are illustrative of the above.

Example 1

15 Four solutions were formed from aliquots, each aliquot presenting an equal initial asparaginase activity (Novozymes A / S), and each aliquot, diluted with distilled water or drinking water so that each solution has a total volume of approximately 50 ml . Drinking water for solutions # 3 and 4 was drinking water supplied by the municipal supply company of North Texas to Plano, TX, USA. The types of water used in each solution are described in Table 1a, below.

20 Table 1a. Types of water used to prepare an asparaginase solution.

Solution No.

Water type

one

Distilled water

2

Distilled water

3

Drinking water from the municipal water supply company of North Texas to Plano, TX, USA

4

Drinking water from the municipal water supply company of North Texas to Plano, TX, USA, mixed with 0.1 M hydrochloric acid added to reach a pH of 6

Each of solutions # 2 to 4 was heated at about 35 ° C for about 40 minutes before

25 of measuring enzymatic activity and pH using solution # 1 as a control for the comparison of residual enzymatic activity. Solution # 1 was refrigerated for about 40 minutes at a temperature of about 10 ° C.

The measured values are shown in Table 1b, below:

30 Table 1b. Residual enzymatic activity of distilled water and drinking water.

Solution No.

pH of the solution Relative activity

one

6.93 100%

2

7.00 103%

3

8.22 38%

4

7.55 48%

It should be noted that the test results of enzyme activity and residual enzyme activity are

35 obtained using the test procedure described at the end of the present exposure. Compared to solution # 1 (control), solution # 2 did not lose enzyme activity. Solution # 3 was slightly alkaline, with a pH of approximately 8.22, and the asparaginase enzyme lost approximately 62% of its activity after approximately 40 minutes at approximately 35 ° C. The addition of diluted hydrochloric acid to drinking water (solution # 4) reduced the pH to approximately 7.55, and asparaginase lost approximately 52% of its

40 activity after about 40 minutes of heating at about 35 ° C. Consequently, apparently the basicity of solution # 3 is responsible for some loss of enzyme activity. It is generally recognized that pH has an impact on asparaginase activity and that asparaginase activity is higher if the pH is between about 4 and about 7.

45 Example 2

Four solutions were prepared from aliquots, each aliquot presenting an equal initial asparaginase activity (Novozymes A / S), and each aliquot, diluted with deionized water or drinking water so that each solution had a total volume of approximately 50 ml. . The types of water used in each are described.

50 solution in Table 2a, below: Table 2a. Asparaginase solutions prepared from water from different supply companies.

Sun No.

Water type

one

Deionized water (control)

2

Drinking water from the municipal water supply company of North Texas in Plano, TX, USA

3

Drinking water supply to residents of Duncanville, TX, USA

4

Water used in a food manufacturing process in Mexicali, Mexico

5 Each of solutions # 2 to 4 was heated at about 35 ° C for approximately 40 minutes before measuring the levels of chlorine, water hardness, pH and enzymatic activity. The control was not heated. The measured values are shown in Table 2b, below.

Table 2b Residual enzymatic and chemical activity of water from three different drinking water solutions 10

Solution No.

Free chlorine (mg / l) Total Chlorine (mg / L) Total hardness (mg / l) pH Activity

one

0 0 0 6.89 100%

2

1.0 1.0 232 7.47 9%

3

0.02 0.02 90 7.87 85%

4

0.02 0.06 28 8.00 89%

These data clearly demonstrate the negative impact that chlorine has on residual enzyme activity. For example, solution # 1 (control) had no chlorine and had the highest residual enzyme activity. Solution # 2 had the lowest level of residual enzyme activity and the highest level of free chlorine and hardness

15 total.

Solution # 3 had a relatively low concentration of free chlorine and a moderate hardness level, with a residual activity exceeding 80%. Solution # 4 had a concentration of free chlorine similar to that of solution # 3 and a lower hardness level, resulting in a slightly higher residual activity. Table 2b

20 demonstrate that the residual enzymatic activity of asparaginase is inversely proportional to the level of chlorine.

Example 3

Four solutions were prepared from aliquots, each aliquot presenting an equal initial activity of

25 asparaginase (Novozymes A / S), and diluting each aliquot with deionized water or drinking water so that each solution had a total volume of approximately 50 ml. The types of water in each sample are presented in Table 3a, below.

Table 3a. Types of chlorinated water used to prepare an asparaginase solution. 30

Sun No.

Water type

one

Deionized water (control)

2

Deionized water + enough hypochlorite to provide 12 ppm of chlorine

3

Drinking water from the municipal water supply company of North Texas in Plano, TX, USA

4

Drinking water from the municipal water supply company of North Texas in Plano, TX, USA, filtered three times through a BRITA filter

Each of solutions # 2 to 4 was heated at about 35 ° C for 40 minutes before measuring the level of free chlorine, total hardness, pH and residual enzymatic activity. 35 The measured values are shown in Table 3b, below: Table 3b. Residual enzymatic activity of solutions that present various levels of chlorine.

Solution No.

Free chlorine (mg / l) Total hardness (mg / l) pH Activity

one

0 - 4.81 100%

2

Not measured - 5.87 4%

3

Not measured 228 7.10 twenty-one%

4

0 twenty 4.99 102%

As Table 3b indicates, above, the addition of chlorine to deionized water as shown in solution # 2,

or the presence of chlorine in drinking water as shown in solution # 3, clearly reduces the residual activity of the asparaginase enzyme. In addition, the removal or absence of chlorine clearly results in an activity

5 increased enzyme, as demonstrated by the level of residual activity of the enzyme in the deionized water of solution # 1 and as demonstrated by the residual enzymatic activity in water filtered by BRITA in solution No. 4. The level of chlorine for the solution No. 2 was not measured because chlorine in the form of sodium hypochlorite had been added to the solution. In addition, in the case of drinking water, it is known that the relative level of chlorine in solution # 3 is similar to the levels in drinking water.

Example 4

The objective of this test was to analyze the effect of chlorine on enzymatic activity by adding the amount of chlorine present in drinking water to deionized water, which does not contain chlorine, in order to

15 determine the effects of chlorine on asparaginase activity.

Four solutions were prepared from aliquots, each aliquot presenting an equal initial activity of asparaginase (Novozymes A / S) and each aliquot was diluted with deionized water or drinking water so that each solution had a total volume of approximately 50 ml. The types of water in each sample are

20 presented in Table 4a, below.

Table 4a. Types of chlorinated water used to prepare an asparaginase solution.

Solution No.

Water type

1 (control)

Deionized water

2

Deionized acidified water with enough sodium hypochlorite to result in water that had 1.2 ppm of chlorine and enough hydrochloric acid to result in an acidic pH

3

Acidified deionized water with enough sodium hypochlorite to result in water that has 0.2 ppm of chlorine and enough hydrochloric acid to result in an acidic pH

4

Drinking water from the municipal water supply company of North Texas in Plano, TX, USA

Each of solutions # 2 to 4 was heated at about 35 ° C for 40 minutes before measuring chlorine, pH and residual enzymatic activity. Solution # 1 was not heated. The measured values are shown in Table 4b, below:

Table 4b Residual enzymatic activity of solutions that presented various levels of chlorine. 30

Solution No.

Free chlorine (mg / l) Total Chlorine (mg / L) pH Activity

one

- - 4.85 100

2

1.2 1.2 4.69 3

3

0.1 0.2 4.62 108

4

0.8 1.1 6.84 14

The data in Table 4b, above, demonstrate that, in the case where only chlorine is added to the water, the residual asparaginase activity is substantially reduced. However, at relatively low levels, chlorine had a lower impact on residual enzyme activity.

Example 5

Five solutions were prepared to determine the potential effects of the modification of drinking water on the residual activity of asparaginase. Each solution was prepared from aliquots, each aliquot presenting a

40 equal initial asparaginase activity (Novozymes A / S), and each aliquot was diluted with deionized water or drinking water so that each solution had a total volume of approximately 50 ml. Citric acid was added so that the solutions were slightly acidic. The types of water used in each solution are described in Table 5a, below:

Table 5a. Drinking Water Modifications

Solution No.

Water type

1 (control)

Deionized water

2

Drinking water from the municipal supply company of North Texas in Plano, TX, USA.

3

Acidified drinking water from the municipal supply company of North Texas in Plano, FX, USA, with enough citric acid to present 100 ppm of citric acid.

4

Drinking water from the municipal water supply company of North Texas in Plano, TX, USA, with enough citric acid to present 100 ppm of citric acid and 950 ppm of EDTA.

5

Acidified drinking water from the municipal supply company of North Texas in Plano, TX, USA, with enough citric acid to present 100 ppm of citric acid and with enough thiosulfate to present 10 ppm of sodium thiosulfate.

Each of solutions # 2 to 5 was heated at about 35 ° C for 40 minutes before measuring free chlorine, total chlorine, pH and residual enzymatic activity. Solution # 1 was not heated. The measured values are shown in Table 5b, below:

Table 5b Residual enzymatic activity of various treated drinking water solutions.

Solution No.

Free chlorine (mg / l) Total Chlorine (mg / L) pH Activity

one

0 0 - 100%

2

0.2 1.2 7.53 12%

3

0.4 0.8 5.81 32%

4

1.0 1.0 6.45 100%

5

0.1 0.4 5.65 86%

Figure 1a is a graphical representation of the residual enzyme activity after various drinking water treatments. The enzymatic activity is represented by the columns in the column chart and the total chlorine concentration is represented by the line (150). As the data show, thiosulfate (added at a level approximately 5 times higher than the concentration of chlorine in drinking water) reduced the concentration of chlorine and increased enzyme activity to 86% (140). Drinking water, which has a total chlorine concentration of 1.2 ppm, had a relatively low residual activity of only 12% (110). Citric acid reduced the level of chlorine in drinking water and increased enzyme activity to 32% (120).

The enzymatic activity (130) in drinking water with EDTA was equivalent to the enzymatic activity of deionized water (100), but EDTA only slightly reduced total chlorine. Without restricting themselves to any particular theory, applicants believe that EDTA could wrap, and thus protect, the enzyme against chlorine, or that EDTA could block chlorine. For example, chlorine is still observed when tested, although the reaction, a reversible reaction between EDTA and chlorine, could prevent the chlorine from oxidizing, or otherwise reacting, with asparaginase. In this way, EDTA apparently inactivates chlorine. Accordingly, in one embodiment, additives can be added that inhibit chlorine from reducing asparaginase activity and / or inactivating chlorine.

Example 6

Five solutions were prepared to determine the potential effects of hard water constituents usually present in drinking water. Each solution was prepared from aliquots, each aliquot presenting an equal initial activity of asparaginase (Novozymes A / S), and each aliquot was diluted with deionized water or drinking water so that each solution had a total volume of approximately 50 ml. . Salt was added to each saline solution to reach a salt concentration of 5 mM (5 millimolar), which is approximately double the concentration of calcium carbonate present in drinking water from Plano, TX. For example, in reference to Table 3b, above, the total hardness of solution # 3 (drinking water from Plano) was 228 mg / l, which corresponds to approximately 2.28 mM. The various types of salts used in each solution are described in Table 6a below:

Table 6a. Salts added to deionized water.

Solution No.

Water type

one

Deionized water

2

Deionized Water + Sodium Chloride

3

Deionized Water + Calcium Chloride

4

Deionized Water + Magnesium Nitrate

5

Deionized Water + Sodium Bicarbonate

Each of solutions # 2 to 5 was heated at about 35 ° C for 40 minutes before measuring residual enzyme activity. The measured values are shown in Table 6b, below:

Table 6b Residual enzymatic activity of various mixtures of salt and water.

Solution

Activity

one

100%

2

99%

3

101%

4

96%

5

102%

Figure 1b is a graphical representation of the residual enzymatic activity of various mixtures of salt and water and

10 graphically show the results of Table 6b, above. The added salt did not show any apparent effect on enzyme stability. Consequently, chlorine is believed to be responsible for most of the loss of asparaginase activity.

Example 7

15 Two aliquots having an equal initial asparaginase activity were diluted with deionized water (cell 1) and running water (cell 2), in order to prepare a first asparaginase solution and a second asparaginase solution. Each solution was kept for 30 minutes at room temperature and then each asparaginase solution was added to corn dough. Asparagine in the mass was measured 5 minutes and

20 10 minutes after the enzyme is added to the dough and the measured values are shown in Table 7, below.

Table 7. Level of asparagine in corn mass using enzyme mixed with drinking water and deionized water.

Type of water used for enzyme dilution

Mass sample Asparagine (ppm)

Deionized water

5 minutes after the addition of the first asparaginase solution 3.6

Deionized water

10 minutes after the addition of the first asparaginase solution 2.9

Drinking water from the municipal supply company of North Texas in Plano, TX, USA

5 minutes after the addition of the second asparaginase solution 37.2

Drinking water from the municipal supply company of North Texas in Plano, TX, USA

10 minutes after the addition of the second asparaginase solution 24.2

The level of asparagine in the corn mass presented in Table 7, above, demonstrates that the resulting level of asparagine is highly dependent on the underlying diluted asparaginase solution. In the embodiment shown above, the level difference was approximately an order of magnitude of the level of asparagine in the corn dough after treatment with deionized water versus treatment with water.

30 drinkable.

The data presented above clearly indicate that the level of active chlorine should be reduced to maximize the residual activity of asparaginase. Because deionized water and distilled water are expensive, the present invention provides a way to maximize residual enzyme activity by eliminating and / or

35 selective inactivation of drinking water chlorine or other water source.

Any method known in the art that can reduce the concentration of enzymatic activity reducing components in drinking water can be used, including, but not limited to, the treatment of drinking water to reduce the concentration of the activity reducing component by water filtration. drinking to

Through activated carbon, an air bubbler (to volatilize chlorine), reverse osmosis systems and / or ion exchange resins. Drinking water can also be treated by mixing drinking water with deionized water or distilled water in sufficient quantities to reduce the concentration of the activity reducing components to prepare a stable enzymatic solution.

45 As used herein, a "hijacker" It is any additive that retains enzymatic activity by reaction with chlorine. Consequently, sequestrants of

enzyme reducing components. For example, in one embodiment, thiosulfate, a chlorine sequestrant, is added to drinking water. In addition, other additives can be used to inactivate chlorine. For example, because chlorine is a strong oxidizing agent, reducing agents can also be added to drinking water to react with chlorine. Reducing agents are known in oxidation-reduction chemistry that are electron donor compounds and oxidizing agents are known to be electron acceptors. Accordingly, in one embodiment, one or more reducing agents (eg electron donors) can be added to a source of drinking water in order to inactivate or neutralize the chlorine. Examples of reducing agents include, but are not limited to, stannous chloride dihydrate, sodium sulphite, sodium metabisulfite, ascorbic acid, derivatives of ascorbic acid, isoascorbic acid (erythorbic acid), salts of derivatives of ascorbic acid, iron, zinc, ferrous ions and combinations. thereof.

In one embodiment, the present invention reduces the concentration of total chlorine to a level between about 0 and a level less than about 0.5 ppm and preferably to a level between 0 and about 0.1 ppm.

In one embodiment, the asparaginase can then be mixed with the treated water to prepare a stable asparaginase solution and then the asparaginase solution can be mixed with the food product. In one embodiment, the drinking water has been sufficiently treated and a stable enzyme or asparaginase solution is achieved in the event that the residual enzyme activity is at least about 80% and more preferably at least about 90% at least 30 minutes and more preferably at least about 4 hours after the addition of the enzyme to the treated drinking water. In one embodiment, the residual enzyme activity is at least about 90% for the time necessary to mix an asparaginase solution in a dough.

From the present discussion, the person skilled in the art will be able to determine and provide the necessary aqueous composition that results in the desired residual enzymatic activity.

The food products to which the asparaginase solution can be added comprise, in a non-limiting manner, masses, suspensions and other consumable products in which it is desired to reduce the level of acrylamide. For example, in one embodiment, the asparaginase solution is added to a potato suspension prepared from potato flakes. In one embodiment, the potato suspension is prepared by adding the asparaginase solution to potato flakes. In one embodiment, the asparaginase solution is used to add to water and is added to a farinaceous composition to prepare a dough. In one embodiment, the asparaginase solution is added to corn dough.

In one embodiment, the present invention comprises a system for providing a stable asparaginase solution that can be added to a food ingredient that has asparagine. In one embodiment, the system comprises a treatment system for treating water. The treatment system can remove components such as chlorine by an activated carbon process or by other disposal procedures indicated above, and / or the treatment system can provide additives that comprise, in a non-limiting manner, reducing agents, chlorine sequestrants or EDTA , which increase the activity of asparaginase to a level that is higher than if the additive has not been added. The treated water can then be directed to a mixing tank in which the asparaginase can be diluted therein to prepare a stable asparaginase solution. Then, the asparaginase solution can be dosed or otherwise added to a dough used to prepare a food manufactured, or thermally processed as indicated above. The dough can then be further processed (for example, formed by extrusion and laminated and thermally processed) as is well known in the art. The person skilled in the art, from the present disclosure, will appreciate that the present invention can be used in any case where an asparaginase solution is desired to reduce the level of acrylamide in a food product.

In one embodiment, the invention comprises a system comprising a source of drinking water and a source of asparaginase, a treatment system that can be operated to increase the activity of asparaginase in the treated water to a level higher than that achieved in the case that the treatment had not been performed, and an administration system that can be operated to mix the treated drinking water and asparaginase. In one embodiment, the administration system comprises a mixing tank that receives treated water from the treatment system and asparaginase.

The test procedure used to determine asparaginase activity for the Examples in the present application is shown below:

I. Background. The SIGMA procedure for asparaginase activity used a Tris buffer at pH 8.6 (Sigma catalog No. A-4887). Because food grade asparaginase exhibits a low activity at pH 8.6, the test pH was modified to pH 7.0 using MOPS (3-morpholinopropanesulfonic acid).

II. Beginning.

III. Conditions: T = 37 ° C, pH = 7.0, A436, light path = 1 cm

IV. Procedure: determination of the spectrophotometric stop rate 5

V. Reagents

to. MOPS 100 mM sodium salt (3-morpholinopropanesulfonic acid). Dosage of 2.09 grams of MOPS (Sigma nº M5162). Dissolution in approximately 60 ml of DI water at room temperature. Addition of

10 sodium hydroxide to adjust the pH to 7.0. Flush to 100 ml with DI water. Storage in a refrigerator when not in use.

b. 189 mM L-asparagine solution. Dosage of 0.25 grams of anhydrous L-asparagine and dissolution in

10 ml of DI water. Storage in the fridge when not in use. After cooling, sonication to dissolve the asparagine crystals prior to use.

C. Standard solution of 6 mM ammonium sulfate ((NH4) 2SO4 standard). Dosage of 0.079 grams of ammonium sulfate in an analytical balance and weight registration to an accuracy of 0.0001 g. Dissolution and flush up to 100 ml volume using DI water. Storage in the fridge when not in use.

d.

 1.5 M trichloroacetic acid (TCA). Dosage of 2.45 grams of trichloroacetic acid. Dissolution and flush to 10 ml with DI water.

and.

 Ammonium color reagent. Test kit for high concentrations of ammonium nitrogen,

25 nesslerization, LaMotte code number 3642-SC, cat. of VWR 34186-1914. Reagent # 2 contains mercury.

F. Asparaginase enzyme solution: immediately before use, prepare a solution containing 2.0 to 4.0 units / ml of asparaginase at room temperature in deionized water. In the case of

30 that the enzyme is frozen, thaw completely in warm water before removing an aliquot for dilution. For typical enzyme concentrations, 0.1 ml of enzyme solution can be diluted to 50 ml.

SAW. Procedure: 35

to.

 Set the heating block for the vials at 37 ° C.

b.

Use an adjustable micropipette to transfer the following reagents to the vials (ml):

C.

 Cover vials and enter the heating block at 37 ° C. Start stirring the heating block.

d.

 Remove the vials from the heating block after 30 minutes. Uncover the vials, immediately add TCA reagent and mix. Next, add reagent F (enzyme solution) to the enzyme blank. For the

Reagent

 Test Enzyme white Standard 1 Standard 2 Standard 3 Reagent blank

A (buffer)

1.00 1.00 1.00 1.00 1.00 1.00

B (L-ASN)

0.10 0.10 --- --- --- ---

C (ammonium standard)

--- --- 0.25 0.50 1.00 ---

DI water

0.90 0.90 0.85 0.60 0.10 1.10

F (enzyme solution)

0.10 --- --- --- --- ---

In enzyme test solutions, the time between the extraction of the heating block vials and the addition of the TCA should be as short as possible. After the addition of the TCA, the time elapsed before the ammonium measurement is not critical. For targets and standards, the time between removal of the heating block and the addition of the TCA is not critical.

Reagent

 Test Enzyme white Standard 1 Standard 2 Standard 3 Reagent blank

D (TCA)

0.10 0.10 0.10 0.10 0.10 0.10

F (enzyme solution)

--- 0.10 --- --- --- ---

and. Pipette 0.20 ml of each solution into the test tubes or vials. Addition of 4.30 ml of deionized water, 4 drops of reagent No. 1 of LaMotte and 0.50 ml of No. 2 of LaMotte. Mix the solution and leave at room temperature for 10 to 20 minutes before reading the absorbance at 436 nm in a 1 cm cell. Set zero

5 of the spectrophotometer with DI water.

VII. Calculation of the results.

F. Enzyme activity is calculated from a calibration curve for ammonia (μmoles / 0.2 ml). 10

g. Description of the calculation stages.

i. Calculation of the concentration of the standard solution of ammonium sulfate:

15 mM = (0.0809 g) * (1,000 mM / M) * (2 NH3 / NH4SO4) / ((132.14 g / mol) * (0.1 l)) = 12.24 mM = mmol / l = μmoles / ml in which 0.0809 grams is the weight of ammonium sulfate for the standard.

ii. Calculation of μmoles of NH3 in 2.2 ml standards: 20

μmoles of NH3 in 2.2 ml = (μmoles of NH3 / ml of standard solution) * (ml of standard)

iii. Calculation of μmoles of NH3 / 0.2 ml: 25 μmoles of NH3 / 0.2 ml = (μmoles of NH3 in 2.2 ml) * (0.2 ml) / (2.2 ml)

iv. Calculation of the regression curve with:

x = A436 30 y = μmoles NH3 / 0.2 ml

v. The μmoles of NH3 / 0.2 ml are calculated from the calibration curve: 35 μmoles of NH3 / 0.2 ml = (slope) * (A436) + Cut-off value with y-axis

saw. The activity of the diluted enzyme solution is calculated using the following formula:

Units / ml of enzyme = (μmoles of NH3 released) * (2.20) / (0.2 * 30 * 0.1) in which:

40 2.20 ml = volume of stage 1 (stage 1 is the enzyme test solution) 0.2 ml = volume of stage 1 used in stage 2 (stage 2 is color development) 30 minutes = test time in minutes 0.1 ml = volume of enzyme used

vii. The dilution factor is 50 ml divided by the volume of concentrated enzyme diluted to 50 ml.

viii Enzyme solution concentration before dilution = (units / ml of diluted solution) * (dilution factor)

Although the invention has been particularly set forth and described with reference to various embodiments, the person skilled in the art will appreciate that various other approaches can be carried out for the conservation of the residual activity of asparaginase in solution without departing from the scope. of the present invention, as defined in the claims.

Claims (11)

1. Procedure for increasing the activity of asparaginase in a solution, said procedure comprising the steps consisting of: 5 a) treating a drinking water to prepare a treated water, wherein said treated water further comprises a chlorine concentration of less than about 0.5 ppm; Y b) mixing the asparaginase with said treated water to prepare an asparaginase solution. 10

2.

Method according to claim 1, which comprises treating the drinking water with an acid.

3.

Method according to claim 1, which comprises treating drinking water using activated carbon.

Method according to claim 1, wherein said water treated in step a) is treated using an ion exchange resin. 5. The method of claim 1, wherein said water treated in step a) is treated using osmosis inverse twenty

6.

Process according to claim 1, wherein said treated water is treated by air bubbling.

7.

Method according to claim 1, wherein said asparaginase solution comprises a residual activity of at least about 80%, wherein optionally the residual activity is at least

About 80% for at least about 30 minutes after the addition of asparaginase to drinking water. 8. A process according to claim 1, wherein said treated water is treated with a reducing agent, wherein said reducing agent optionally comprises one or more agents selected from stannous chloride 30 dihydrate, sodium sulphite, sodium metabisulfite, ascorbic acid, derivatives of ascorbic acid, isoascorbic acid (erythorbic acid), salts of derivatives of ascorbic acid, iron, zinc, ferrous ions and combinations thereof. 9. The method according to claim 1, wherein said treated water comprises a pH lower than approximately 8.0. 35 Method according to claim 1, wherein said drinking water is treated with an additive sufficient to reduce the final level of chlorine to a level that is lower than if the additive in which said treated water is optionally not added it deals with a chlorine sequestrant, or in which said additive optionally comprises thiosulfate. 11. System comprising: a source of drinking water; 45 a source of asparaginase; a treatment system that can be operated to treat drinking water before mixing with asparaginase; Y 50 a supply system that can be operated to mix the treated drinking water and asparaginase. 12. System according to claim 11, wherein said treatment system eliminates chlorine or inactivates chlorine.